Animal model of diabetic keratopathy

Diabetic keratopathy is one of the most common ocular complications in diabetes mellitus. Protocol for diabetes induction in rat model also has been established in many centers. Nonetheless, method in developing diabetic keratopathy rat model has not been well covered. Streptozotocin (STZ)-induced diabetes is widely being used as animal diabetic model. The purpose of this study is to obtain an animal model of diabetic keratopathy that can be used to study the morphology, metabolism, and function of cornea in cases where human samples can be difficult to obtain.A single dose STZ (50 mg/kg) was injected intraperitoneally to control and intervention group. Plasma glucose level concentrations were tested in day 3 post injections. Obtained animal model of diabetic keratopathy, with significant difference of blood glucose level between intervention and control group (P<0.00). Sensibility of cornea was decreased by week 14 th in intervention group. Epithelial defect were more prominent in diabetic group. Despite the differences between human and animal characteristic of diabetic keratopathy, the use of animal models has contributed to better understanding of this disease and to examine more effective treatment.


■ INTRODUCION
The increasing number of diabetes case in Indonesia is accompanied by increasing complication on the eyes, which leads to vision loss (Wild et al. 2004). Of which, diabetic keratopathy is one of the most common ocular complication in diabetes. Diabetic keratopathy is a disease of the cornea characterized by impaired morphology, metabolism, and function of cornea. Dry eye syndrome and impaired wound healing in diabetic patients also leads to diabetic keratopathy (Yin et al. 2011).
Interaction of glucose and reactive oxygen species in diabetic patients leads to increasing extracellular osmotic stress. This oxidative stress affects mitochondria permeability, activate apoptosis of caspase cells, and induce apoptosis of neuron and Schwann cells. Neuronal apoptosis leads to irreversible nerve damage (Veira-Potter et al. 2016).
Streptozotocin (STZ)-induced diabetes is widely being used as animal diabetic model. Its mechanism in selective destruction of pancreatic B cell is effectively creating insulin deficiency, hypeprglycemia, as well as polydipsia and polyuria state (King 2012). Two protocols used to produce STZ-induced diabetic rat including multiple administrations of low-dose STZ and administration using a single, high dose STZ (Ljubimov 2017).
Protocol for induction of diabetes rat model also has been carried out in many centers. Nonetheless, studies and protocol in developing diabetic keratopathy rat model has not been well covered. The aim of this paper is to share our experience in developing rat model of diabetic keratopathy.

■ MATERIALS AND METHODS
Animal care: Male Sprague-Dawley rats, 3-4 months of age, weighing 250-350 gram were used for the studies. They were housed in 30x40x20 cm cage with ambient temperature of 25-30 o C, relative humidity of 80-90%, and stable light.

Injection of STZ:
Before injection, animals were tested for plasma glucose concentration. They were divided randomly into intervention and control group. A single dose STZ (50 mg/kg) diluted with fresh citric acid solution (0.01M pH 4.5) was injected intraperitoneally. Control group were injected with citric acid buffer. After injection, animals had free access to food. On day 3 post-STZ injection, tail-vein blood sample were drawn. Animals are considered suitable for study when blood glucose level of STZ-injected rat is ≥250 mg/dl. If in 3 days glucose level has not reached 250 mg/dl, rats were given second dose of STZ. Insulin was given daily to prevent adverse effect of severe hyperglycemia.
Examination of diabetic keratopathy rat models: On 10 th day post-STZ injection, animals were examined using slit lamp bio microscope to assess cornea integrity, irregularity, and epithelial defect. Before examination a single drop of fluorescein eye drop were administered. Sensibility of cornea was assessed using Cochet-Bonnet aesthesiometer.

■ RESULTS AND DISCUSSION
During first week post-STZ induction, average of blood glucose level in diabetic group was 322±26 mg/dl, compared to normal group 91±1 mg/dl (Figure 1). Normal group revealed stable concentration of blood sugar of 89-96 mg/dL, meanwhile in intervention group using STZinduction, blood sugar level were 266-365 mg/dL (P<0.00). Clinical examination on keratopathy eyes were conducted on 10 th and 14 th week post-STZ injection (Figure 2). In diabetic group, there was a significant decrease in cornea sensibility from 55.25±1.7 mm in week 10 th into 42.75±1.92 mm in week14 th (P=0.012). Meanwhile in control group, sensibility of cornea was unremarkable from 59.33±1.21 mm in 10 th week into 59.17±1.21 mm in 14 th week. Cornea epithelial defect was examined using fluorescein and slit lamp. Corneal lesion scoring were used to classify degree of lesion, diabetic group showed all subjects suffered from dendritic ulceration in week 14 th , while in control group 4 subjects had normal epithelial and 2 subjects had punctate ulceration. Cornea lesion was found getting worse in week 14 th compared to week 10 th in diabetic model. The significant difference of blood sugar level between intervention and control groups shows the success of diabetes induction by streptozotocin in rats. Our diabetic rats blood glucose level was ranging from 266-365 mg/dL, whereas control rats was 89-96 mg/dL (P<0.00). Significant difference between STZ-treatment and control group were the major determinant of diabetes induction success (Furman 2015).
Our study revealed a significant decrease of corneal sensitivity from week 10 th compared with week 14 th , suggesting potential damage of sensory neurons with constant hyperglycemia. Other study revealed decrease of corneal sensitivity in 8 th week (Yin et al. 2011, Bikbova et al. 2016. Reduced innervation was prominent in diabetic rats with the reduction of long nerve fibers was documented (Cousen et al. 2007). Reduced innervation also contributing to lacrimal gland function, thus reducing tear protection and contributing to dry eye (Cousen et al. 2007).
Integration of cell junction have vital role in the formation and maintenance of epithelial barrier of epithelia, punctate keratitis is observed in some diabetic patients (Yin et al. 2011). Our study found more severe corneal lesion in diabetic group, including punctate and dendritic keratitis.

■ CONCLUSION
Diabetic keratopathy can lead to morbidity and eventually vision loss. Therefore, model of diabetic keratopathy have been developed in animals to analyze the disease. Despite the differences between human and animal characteristic of diabetic keratopathy, the use of animal models has contributed to better understanding of this disease and to examine more effective treatment.